Rubisco activase with increased thermostability and methods of use thereof

ABSTRACT

The present invention provides thermostable polypeptides related to  Arabidopsis  Rubisco Activase polypeptides. Nucleic acids encoding the polypeptides of the invention are also provided. Methods for using the polypeptides and nuclei acids of the invention to enhance resistance of plants to heat stress are encompassed.

CROSS-REFERENCE TO RELATED APPLICATION

This utility application is a divisional application claiming benefitfrom U.S. Utility patent application Ser. No. 12/474,298 filed May 29,2009 which is a divisional of U.S. Utility patent application Ser. No.11/867,723, filed Oct. 5, 2007, now granted U.S. Pat. No. 7,557,267issued Jul. 7, 2009, which is a divisional of U.S. Utility patentapplication Ser. No. 11/507,729, filed Aug. 22, 2006, now granted U.S.Pat. No. 7,314,975 issued Jan. 1, 2008, which also claims the benefitU.S. Provisional Patent Application Ser. No. 60/711,449, filed Aug. 24,2005 and U.S. Provisional Serial Application No. 60/733,110, filed Nov.2, 2005, all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to increasing the levels ofphotosynthesis in plants grown under increased temperatures. Moreparticularly, the present invention relates to improving thethermostability of the photosynthetic enzyme Rubisco Activase.

BACKGROUND OF THE INVENTION

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is animportant enzyme in the photosynthetic process. This enzyme incorporatesCO₂ into plants during photosynthesis. Atmospheric oxygen competes withCO₂ as a substrate for Rubisco, giving rise to photorespiration andmaking Rubisco the rate-limiting step in photosynthesis.

Rubisco Activase (RCA) is a protein that catalyzes the activation ofRubisco, which in turn, regulates photosynthesis by initiatingphotosynthetic carbon reduction and photorespiratory carbon oxidation.The Rubisco Activase enzyme catalyzes the release ofribulose-1,5-bisphosphate (RuBP) from Rubisco. This newly unoccupiedsite on Rubisco is now free to bind the CO₂ and Mg²⁺ activators in orderfor photosynthesis to proceed. Rubisco Activase is also responsible forreleasing sugar phosphate inhibitors from Rubisco and restores Rubiscocatalytic activity. Thus, if Rubisco Activase is impaired, Rubiscoremains inactive and photosynthesis slows.

Rubisco Activase is thermo-labile and thus has decreasing activity withincreasing temperatures. As a result, the photosynthetic process slowsdue to the lack of Rubisco activation. The Rubisco Activase enzymedenatures under increased temperatures, thus rendering the enzyme unableto convert inactive Rubisco to the active form. Arabidopsis contains twoRCA isoforms, the short thermolabile (RCA1) and the long relativelythermostable (RCA2) forms that are generated by alternative splicing ofpre-mRNA (Werneke, et al., (1989) Plant Cell 1:815-825).

Crop plants grown in hot climates could benefit from increasingphotosynthetic levels. Accordingly, if the rate limiting step inphotosynthesis could be made more heat tolerant, crop plants could moreeasily grow in these climates.

SUMMARY OF THE INVENTION

The present invention relates to Rubisco Activase derived polypeptidesthat are more thermostable than naturally occurring Rubisco Activase.The Rubisco Activase derived polypeptides of the invention substantiallyretain activity in plants grown under conditions of increasedtemperature. Nucleic acid molecules encoding the polypeptides of theinvention are also encompassed.

In addition to the Rubisco Activase derived polypeptides of theinvention, it will be appreciated that the invention also encompassesvariants thereof, including, but not limited to, any substantiallysimilar sequence, any fragment, analog, homolog, mutant or modifiedpolypeptide thereof. The variants encompassed by the invention are atleast partially functionally active (i.e., they are capable ofdisplaying one or more known functional activities associated with wildtype Rubisco Activase) under heated conditions. Nucleic acid moleculesencoding the variant polypeptides are also encompassed.

Vectors comprising one or more nucleic acids of the invention are alsoencompassed.

Cells comprising a polypeptide, nucleic acid molecule and/or vector ofthe invention are also encompassed.

The present invention also relates to transgenic plants comprising apolypeptide, nucleic acid molecule, and/or vector of the invention. Thetransgenic plants can express the transgene in any way known in the artincluding, but not limited to, constitutive expression, developmentallyregulated expression, tissue specific expression, etc. Seed obtainedfrom a transgenic plant of the invention is also encompassed.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C: Characterization of wild type RCA (RCA1) and thermostablevariants (183H12, 301C7 and 382D8). (A) Rubisco activity afteractivation by activase after treatment at 25° C. (white), 40° C. (gray)and 45° C. (black). Activase proteins were incubated at the indicatedtemperatures for 15 min prior to assay at 25° C. (B) Activation ofRubisco under catalytic conditions at 25° C. (white) and 40° C. (gray).(C) ATPase activity of activase proteins incubated at the indicatedtemperatures for 15 min prior to assay at 25° C.

FIGS. 2A-2D: Characterization of the Rubisco Activase mutant (Δrca) atambient CO₂. (A) Immuno blot analysis from leaves of Arabidopsiswild-type (RCA/RCA), heterozygous (RCA/Δrca) and homozygous (Δrca/Δrca)plants. The blot was immunodecorated with polyclonal antibodies raisedagainst the recombinant Arabidopsis RCA1. (B) Photosynthetic performanceof three-week old wild-type (upper) and Δrca (lower) plants as measuredusing fluorescence image analysis. (C) Leaf area of the plants describedin (B) (50 plants per phenotype) at the indicted age. (D) Photograph ofeight week old wild-type (upper) and Δrca (lower) plants.

FIGS. 3A-3C: Molecular characterization of Δrca mutant. (A) Schematicmap of the wild-type (RCA) and deletion (Δrca) alleles. Numbers indicatethe RCA exons. The forward and reverse primers for amplification of the1.4 kb PCR products of RCA (RCAf and RCAr) and Δrca (rcaf and rcar)alleles are indicated. (B) PCR analysis (RCA primers-upper panel; rcaprimers-bottom panel) of T1 plants expressing 183H12. Lines number (up)and genetic background (down) are indicated. (C) Western blot analysisof total protein (5 μg/lane) from leaves of the lines described in (B).The blot was probed with polyclonal antibodies raised against therecombinant RCA1.

FIGS. 4A-4D: Functional complementation of Δrca mutants expressing RCA1and thermostable variants 183H12, 301C7 and 382D8 under normal growthconditions (22° C.). Numbers indicate the line designations ofindependent transformation events. (A) Immuno blot analysis of totalprotein from three-week old leaves. (B) Photographs depicting thesimilar size of all the plants described above when grown under normalconditions. (C) Photosynthetic performance of the plants (8 to 10plants/independent line) described above monitored by fluorescence imageanalysis. (D) Effect of temporary (1 hr) moderate heat stress treatmenton photosynthetic rates of Δrca transgenic lines expressing RCA1 andthermostable variants. The net photosynthesis of four independent plantsper line was monitored using an infrared gas analyzer at 22° C. (white)and 30° C. (gray).

FIGS. 5A-5F: Effect of moderate heat stress (30° C. for 4 hr per day) onwild type plants and Δrca mutants expressing RCA1 or thermostablevariants 183H12, 301C7 and 382D8. (A) Photograph of the plants showingdifferential growth rates mediated by the RCA variant. (B) Leaf area of8-10 independent plants per line, analyzed using a fluorescence imageanalysis system. Means followed by common letters are not significantlydifferent at P=0.05 using a protected LSD. (C) Net photosynthesis offour independent plants from selected lines, monitored by gas exchangeanalysis after 2 hr at 30° C. (D) Photograph of mature plants (8-weeksold) described in (C). (E) Number of siliques per plant. Eight to tenindependent plants per line were analyzed. (F) Seed weight/1000 seeds ofseeds harvested from the selected lines (5 independent plants per line).

FIGS. 6A-6D: Effect of 26° C. heat stress on development and yield ofwild type plants and Δrca mutant lines expressing RCA1 and thermostablevariant 183H12. (A) Number of siliques per plant. Ten to twelveindependent plants per line were analyzed. (B) Photograph of siliquesfrom selected lines grown at the indicated temperature showing variationin siliqe size and seed set. Bar=0.5 cm. (C) Seed weight/1000 seeds forseeds harvested from the plants described in (A). (D) Germination rates(at 22° C.) of seeds (250 seeds per line) harvested from Arabidopsisplants that were grown at 22° C. (white) or 26° C. (gray).

DETAILED DESCRIPTION

The present invention provides polypeptides derived from RubiscoActivase. Nucleic acid molecules encoding the polypeptides of theinvention are also provided. Methods for using the polypeptides andnucleic acids of the invention to increase the heat-tolerance of plantscomprising enhancing the thermostability of Rubisco Activase areencompassed.

Polypeptides of the Invention

The present invention relates to Rubisco Activase derived polypeptidesthat are more thermostable than naturally occurring Rubisco Activase. Inpreferred embodiments, the Rubisco Activase derived polypeptide is anyof SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22. Polypeptides ofthe invention also encompass those polypeptides that are encoded by anyRubisco Activase derived nucleic acid of the invention.

In addition to the Rubisco Activase derived polypeptides of theinvention, it will be appreciated that the invention also encompassesvariants thereof, including, but not limited to, any substantiallysimilar sequence, any fusion polypeptide, any fragment, analog, homolog,mutant or modified polypeptide thereof. The variants encompassed by theinvention are at least partially functionally active (i.e., they arecapable of displaying one or more known functional activities associatedwith wild type Rubisco Activase) under heated conditions. Suchfunctional activities include, but are not limited to, biologicalactivities, such as activation of Rubisco; antigenicity, i.e., anability to bind or compete with wild type Rubisco Activase (including,but not limited to, SEQ ID NO: 2) for binding to an anti-RubiscoActivase antibody; immunogenicity, i.e., an ability to generate antibodywhich binds to a wild type Rubisco Activase polypeptide. In preferredembodiments, the variants have at least one functional activity that issubstantially similar to or better than its parent polypeptide (i.e.,the unaltered Rubisco Activase derived polypeptide). As used herein, thefunctional activity of the variant will be considered “substantiallysimilar” to its parent polypeptide if it is within one standarddeviation of the parent.

In one embodiment, polypeptides that have at least one functionalactivity of Rubisco Activase (e.g., Rubisco activation) under heatedconditions and are at least 85%, 90%, 95%, 97%, 98% or 99% identical tothe polypeptide sequence of any of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16,18, 20 and 22 are encompassed by the invention. In specific embodiments,such polypeptides of the invention are altered at one or more, two ormore, five or more, or seven or more positions corresponding to residues42, 130, 131, 168, 257, 274, 293 and 310 of SEQ ID NO: 2 upon optimalalignment of the polypeptide sequence with SEQ ID NO: 2. With respect toan amino acid sequence that is optimally aligned with a referencesequence, an amino acid “corresponds” to the position in the referencesequence with which the residue is paired in the alignment.

As used herein, where a sequence is defined as being “at least X %identical” to a reference sequence, e.g., “a polypeptide at least 95%identical to SEQ ID NO: 4,” it is to be understood that “X% identical”refers to absolute percent identity, unless otherwise indicated. Theterm “absolute percent identity” refers to a percentage of sequenceidentity determined by scoring identical amino acids or nucleic acids asone and any substitution as zero, regardless of the similarity ofmismatched amino acids or nucleic acids. In a typical sequence alignmentthe “absolute percent identity” of two sequences is presented as apercentage of amino acid or nucleic acid “identities.” In cases where anoptimal alignment of two sequences requires the insertion of a gap inone or both of the sequences, an amino acid residue in one sequence thataligns with a gap in the other sequence is counted as a mismatch forpurposes of determining percent identity. Gaps can be internal orexternal, i.e., a truncation. Absolute percent identity can be readilydetermined using, for example, the Clustal W program, version 1.8, June1999, using default parameters (Thompson, et al., (1994) Nucleic AcidsResearch 22:4673-4680).

In another embodiment, fusion polypeptides comprising a Rubisco Activasederived polypeptide or variant thereof are encompassed by the invention.In a specific embodiment, a peptide (such as those disclosed in U.S.patent application Ser. No. 11/150,054) is added onto a polypeptide ofthe invention, whereby the peptide directs localization of the attachedpolypeptide to the plant plastids or the plant photosynthetic organs.

In another embodiment, fragments of Rubisco Activase derivedpolypeptides are encompassed by the invention. Polypeptides areencompassed that have at least one functional activity (e.g., Rubiscoactivation) of Rubisco Activase under heated conditions and are at least25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375or 380 contiguous amino acids in length of any of SEQ ID NOS: 4, 6, 8,10, 12, 14, 16, 18, and 22. In preferred embodiments, the polynucleotidethat encodes the fragment polypeptide hybridizes under stringentconditions to the nucleic acid that encodes any of SEQ ID NOS: 4, 6, 8,10, 12, 14, 16, 18, 20 and 22.

In a specific embodiment, a fragment of the invention corresponds to afunctional domain of Rubisco Activase including, but not limited to, theATP binding domain and the substrate interaction domain (see, e.g., Li,et al., (2005) J Biol Chem 280:24864-24869; Salvucci, et al., (1994)Biochemistry 33:14879-14886 and van de Loo, et al., (1996) Biochemistry35:8143-8148).

In another embodiment, analog polypeptides are encompassed by theinvention. Analog polypeptides may possess residues that have beenmodified, i.e., by the covalent attachment of any type of molecule tothe Rubisco Activase derived polypeptides. For example, but not by wayof limitation, an analog polypeptide of the invention may be modified,e.g., by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. An analog polypeptide of the invention may be modified by chemicalmodifications using techniques known to those of skill in the art,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Furthermore, ananalog of a polypeptide of the invention may contain one or morenon-classical amino acids.

In another embodiment of the invention, a Rubisco Activase derivedpolypeptide is not SEQ ID NO: 2. In yet another embodiment of theinvention, a Rubisco Activase derived polypeptide is not a naturallyoccurring wild type polypeptide.

Methods of production of the polypeptides of the invention, e.g., byrecombinant means, are also provided.

Nucleic Acid Molecules of the Invention

The present invention also relates to the nucleic acid moleculesencoding Rubisco Activase derived polypeptides. In preferredembodiments, the Rubisco Activase derived nucleic acid molecule is anyof SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21. Nucleic acidmolecules of the invention also encompass those nucleic acid moleculesthat encode any Rubisco Activase derived polypeptides of the invention.

In addition to the nucleic acid molecules encoding Rubisco Activasederived polypeptides, it will be appreciated that nucleic acid moleculesof the invention also encompass those encoding polypeptides that arevariants of Rubisco Activase derived polypeptides, including, but notlimited to any substantially similar sequence, any fusion polypeptide,any fragment, analog, homolog, mutant or modified polypeptide thereof.The nucleic acid molecule variants encompassed by the invention encodepolypeptides that are at least partially functionally active (i.e., theyare capable of displaying one or more known functional activitiesassociated with wild type Rubisco Activase) under heated conditions.

In one embodiment, nucleic acid molecules that are at least 70%, 75%,80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any of the nucleic acidmolecules of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 areencompassed by the invention. In specific embodiments, such nucleic acidmolecules of the invention encode polypeptides that are altered at oneor more, two or more, five or more, or seven or more positionscorresponding to residues 42, 130, 131, 168, 257, 274, 293 and 310 ofSEQ ID NO: 2 upon optimal alignment of the nucleotide sequence with SEQID NO: 2.

To determine the percent identity of two nucleic acid molecules, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of a first nucleic acid molecule for optimalalignment with a second or nucleic acid molecule). The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions ×100%). In one embodiment, the twosequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (Karlin and Altschul, (1990) Proc.Natl. Acad. Sci. 87:2264-2268, modified as in Karlin and Altschul,(1993) Proc. Natl. Acad. Sci. 90:5873-5877). Such an algorithm isincorporated into the NBLAST and XBLAST programs (Altschul, et al.,(1990) J. Mol. Biol. 215:403 and Altschul, et al., (1997) Nucleic AcidRes. 25:3389-3402). Software for performing BLAST analyses is publiclyavailable, e.g., through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul, et al., supra). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Forpolypeptides, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=⁻4 and acomparison of both strands. For polypeptides, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10 and theBLOSUM62 scoring matrix (see, Henikoff and Henikoff, (1989) PNAS89:10915).

The Clustal V method of alignment can also be used to determine percentidentity (Higgins and Sharp, (1989) CABIOS 5:151-153) and found in theMegalign program of the LASERGENE bioinformatics computing suite(DNASTAR Inc., Madison, Wis.). The “default parameters” are theparameters pre-set by the manufacturer of the program and for multiplealignments they correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10,while for pairwise alignments they are KTUPLE 1, GAP PENALTY=3, WINDOW=5and DIAGONALS SAVED=5. After alignment of the sequences, using theClustal V program, it is possible to obtain a “percent identity” byviewing the “sequence distances” table on the same program.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

In another embodiment, fragments of Rubisco Activase derived nucleicacid molecules are encompassed by the invention. Nucleic acid moleculesare encompassed that have at least one functional activity of RubiscoActivase (e.g., Rubisco activation) under heated conditions and are atleast 100, 250, 500, 750, 950, 1000 or 1100 contiguous nucleotides inlength of any of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21.

In a specific embodiment, a fragment of the invention corresponds to anucleic acid molecule that encodes a functional domain of RubiscoActivase including, but not limited to, the ATP binding domain and thesubstrate interaction domain (see, e.g., Li, et al., (2005) J Biol Chem280:24864-24869; Salvucci, et al., (1994) Biochemistry 33:14879-14886and van de Loo, et al., (1996) Biochemistry 35:8143-8148).

In another embodiment, a nucleic acid molecule that hybridizes understringent conditions to any one of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15,17, 19 and 21 is encompassed by the invention. The phrase “stringentconditions” refers to hybridization conditions under which a nucleicacid will hybridize to its target nucleic acid, typically in a complexmixture of nucleic acid, but to essentially no other nucleic acids.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer nucleic acids hybridize specifically athigher temperatures. Extensive guides to the hybridization of nucleicacids can be found in the art (e.g., Tijssen, Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Probes, “Overview ofprinciples of hybridization and the strategy of nucleic acid assays”(1993)). Generally, highly stringent conditions are selected to be about5-10° C. lower than the thermal melting point (T_(m)) for the specificnucleic acid at a defined ionic strength pH. Low stringency conditionsare generally selected to be about 15-30° C. below the T_(m). The T_(m)is the temperature (under defined ionic strength, pH, and nucleic acidconcentration) at which 50% of the probes complementary to the targethybridize to the target nucleic acid at equilibrium (as the targetnucleic acids are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Hybridization conditions are typically thosein which the salt concentration is less than about 1.0 M sodium ion,typically about 0.01 to 1.0 M sodium ion concentration (or other salts)at pH 7.0 to 8.3 and the temperature is at least about 30° C. for shortprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longprobes (e.g., greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, and preferably 10 times backgroundhybridization. The phrase “specifically hybridizes” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

In another embodiment of the invention, a Rubisco Activase derivednucleic acid molecule is not SEQ ID NO: 1. In yet another embodiment ofthe invention, a Rubisco Activase derived nucleic acid molecule is not anaturally occurring wild type nucleic acid molecule.

Vectors comprising nucleic acid molecules of the invention are alsoencompassed. Cells or plants comprising the vectors of the invention arealso encompassed.

The term “nucleic acid molecule” herein refers to a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesread from the 5′ to the 3′ end. It includes chromosomal DNA,self-replicating plasmids and DNA or RNA that performs a primarilystructural role.

Rubisco Activase-Derived Sequences

Rubisco Activase derived polypeptides and nucleic acid molecules of theinvention can be created by introducing one or more residuesubstitutions, additions and/or deletions into a wild type (wt) RubiscoActivase (including, but not limited to, Arabidopsis Rubisco Activase(SEQ ID NO: 2)). Generally, Rubisco Activase derived polypeptides arecreated in order to accentuate a desirable characteristic or reduce anundesirable characteristic of a wild type Rubisco Activase polypeptide.In one embodiment, Rubisco Activase derived polypeptides have improvedthermostability over wild type Rubisco Activase. In another embodiment,Rubisco Activase derived polypeptides have enzymatic activity underheated conditions (e.g., 40° C.) that is similar to or higher than theenzyme activity of wild type Rubisco Activase under normal conditions(e.g., 25° C.).

In one embodiment, a wild type Rubisco Activase nucleic acid molecule(e.g., SEQ ID NO: 1) is used as a template to create Rubisco Activasederived nucleic acid molecules. In some embodiments, nucleic acidresidues that encode one or more amino acid residues corresponding toresidues 42, 130, 131, 168, 257, 274, 293 and 310 of SEQ ID NO: 2 uponoptimal alignment of the nucleotide sequence with SEQ ID NO: 2 arealtered such that the encoded amino acid is altered.

Sequence alterations can be introduced by standard techniques such asdirected molecular evolution techniques e.g., DNA shuffling methods(see, e.g., Christians, et al., (1999) Nature Biotechnology 17:259-264;Crameri, et al., (1998) Nature 391:288-291; Crameri, et al., (1997)Nature Biotechnology 15:436-438; Crameri, et al., (1996) NatureBiotechnology 14:315-319; Stemmer, (1994) Nature 370:389-391; Stemmer,et al., (1994) Proc. Natl. Acad. Sci. 91:10747-10751; U.S. Pat. Nos.5,605,793; 6,117,679; 6,132,970; 5,939,250; 5,965,408; 6,171,820;International Publication Numbers WO 95/22625; WO 97/0078; WO 97/35966;WO 98/27230; WO 00/42651 and WO 01/75767); site directed mutagenesis(see, e.g., Kunkel, (1985) Proc. Natl. Acad. Sci. 82:488-492; Oliphant,et al., (1986) Gene 44:177-183); oligonucleotide-directed mutagenesis(see, e.g., Reidhaar-Olson, et al., (1988) Science 241:53-57); chemicalmutagenesis (see, e.g., Eckert, et al., (1987) Mutat. Res. 178:1-10);error prone PCR (see, e.g., Caldwell and Joyce, (1992) PCR MethodsApplic. 2:28-33) and cassette mutagenesis (see, e.g., Arkin, et al.,(1992) Proc. Natl. Acad. Sci., 89:7871-7815); (see generally, e.g.,Arnold, (1993) Curr. Opinion Biotechnol. 4:450-455; Ling, et al., (1997)Anal. Biochem., 254(2):157-78; Dale, et al., (1996) Methods Mol. Biol.57:369-74; Smith, (1985) Ann. Rev. Genet. 19:423-462; Botstein, et al.,(1985) Science, 229:1193-1201; Carter, (1986) Biochem. J. 237:1-7;Kramer, et al., (1984) Cell 38:879-887; Wells, et al., (1985) Gene34:315-323; Minshull, et al., (1999) Current Opinion in Chemical Biology3:284-290).

In one embodiment, DNA shuffling is used to create Rubisco Activasederived nucleic acid molecules. DNA shuffling can be accomplished invitro, in vivo, in silico or a combination thereof. In silico methods ofrecombination can be affected in which genetic algorithms are used in acomputer to recombine sequence strings which correspond to homologous(or even non-homologous) nucleic acids. The resulting recombinedsequence strings are optionally converted into nucleic acids bysynthesis of nucleic acids which correspond to the recombined sequences,e.g., in concert with oligonucleotide synthesis gene reassemblytechniques. This approach can generate random, partially random ordesigned alterations. Many details regarding in silico recombination,including the use of genetic algorithms, genetic operators and the likein computer systems, combined with generation of corresponding nucleicacids as well as combinations of designed nucleic acids (e.g., based oncross-over site selection) as well as designed, pseudo-random or randomrecombination methods are described in the art (see, e.g., InternationalPublication Numbers WO 00/42560 and WO 00/42559).

In another embodiment, targeted mutagenesis is used to create RubiscoActivase derived nucleic acid molecules by choosing particularnucleotide sequences or positions of the wild type Rubisco Activase foralteration. Such targeted mutations can be introduced at any position inthe nucleic acid and can be conservative or non-conservative.

A “non-conservative amino acid substitution” is one in which the aminoacid residue is replaced with an amino acid residue having a dissimilarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid, asparagine, glutamine),uncharged polar side chains (e.g., glycine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan), β-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Alternatively or in addition to non-conservative amino acid residuesubstitutions, such targeted mutations can be conservative. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Following mutagenesis, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined.

In some embodiments, substitutions can be made such that the amino acidat position 42 has an uncharged polar side chain or a β-branched sidechain, at position 130 has a basic side chain, at position 131 has anonpolar side chain or a β-branched side chain, at position 168 has abasic side chain, at position 257 has a nonpolar side chain or aβ-branched side chain, at position 274 has a basic side chain, atposition 293 has a basic side chain and/or at position 310 has an acidicside chain. The amino acid positions can be determined by optimallyaligning the amino acid sequence of the encoded Rubisco Activase derivedpolypeptide with SEQ ID NO: 2.

In another embodiment, random mutagenesis is used to create RubiscoActivase derived nucleic acid molecules. Mutations can be introducedrandomly along all or part of the coding sequence (e.g., by saturationmutagenesis). In certain embodiments, nucleotide sequences encodingother related polypeptides that have similar domains, structural motifs,active sites, or that align with a portion of wild type Rubisco Activasewith mismatches or imperfect matches, can be used in the mutagenesisprocess to generate diversity of sequences.

It should be understood that for each mutagenesis step in some of thetechniques mentioned above, a number of iterative cycles of any or allof the steps may be performed to optimize the diversity of sequences.The above-described methods can be used in combination in any desiredorder. In many instances, the methods result in a pool of alterednucleic acid sequences or a pool of recombinant host cells comprisingaltered nucleic acid sequences. The altered nucleic acid sequences orhost cells expressing an altered nucleic acid sequence with the desiredcharacteristics can be identified by screening with one or more assaysknown in the art. The assays may be carried out under conditions thatselect for polypeptides possessing the desired physical or chemicalcharacteristics. The alterations in the nucleic acid sequence can bedetermined by sequencing the nucleic acid molecule encoding the alteredpolypeptide in the clones.

Additionally, Rubisco Activase derived nucleic acid molecules can becodon optimized, either wholly or in part. Because any one amino acid(except for methionine) is encoded by a number of codons (Table 1), thesequence of the nucleic acid molecule may be changed without changingthe encoded amino acid. Codon optimization is when one or more codonsare altered at the nucleic acid level to coincide with or betterapproximate the codon usage of a particular host. The frequency ofpreferred codon usage exhibited by a host cell can be calculated byaveraging frequency of preferred codon usage in a large number of genesexpressed by the host cell. This analysis may be limited to genes thatare highly expressed by the host cell. U.S. Pat. No. 5,824,864, forexample, provides the frequency of codon usage by highly expressed genesexhibited by dicotyledonous plants and monocotyledonous plants. Thosehaving ordinary skill in the art will recognize that tables and otherreferences providing preference information for a wide range oforganisms are available in the art.

Methods of Assaying Rubisco Activase Activity

The present invention is directed to Rubisco Activase derivedpolypeptides with improved thermostability as compared to wild typeRubisco Activase. As used herein, the term “improved thermostability”refers to the increased ability of Rubisco Activase to activate Rubiscounder heated conditions as compared to wild type Rubisco Activase. Inone embodiment, Rubisco Activase derived polypeptides have enzymaticactivity under heated conditions (e.g., 35° C. or higher) that isgreater than the enzymatic activity of wild type Rubisco Activase underheated conditions. In another embodiment, Rubisco Activase derivedpolypeptides have enzymatic activity under heated conditions (e.g., 26°C. or higher, more preferably 40° C. for in vitro assays) that issubstantially similar to or higher than the enzyme activity of wild typeRubisco Activase under normal conditions (e.g., 20-25° C., morepreferably 25° C. for in vitro assays and 22° C. for in vivo assays). Asused herein, the term “substantially similar” refers to enzymaticactivity of a Rubisco Activase derived polypeptide that is within onestandard deviation of that of wild type Rubisco.

Any method known in the art can be used to assay the activity of RubiscoActivase derived polypeptides (including, but not limited to, Rubiscoactivation and ATP hydrolysis) under heated conditions.

In some embodiments, Rubisco Activase derived polypeptide activity isassayed in vitro. In one embodiment, Rubisco Activase derivedpolypeptides can be assayed for their ability to activate Rubisco whenincubated in a solution comprising deactivated Rubisco, RuBP, ATP and asource of labeled carbon (e.g., [C¹⁴]NaHCO₃). Incorporation of labeledcarbon into 3-phosphoglyceric acid (GPA) can be monitored as anindication of Rubisco activation. In another embodiment, RubiscoActivase derived polypeptides can be assayed for their ability tohydrolyze ATP when incubated in a solution comprising ATP. The assayscan be conducted under heated conditions or under normal conditionsafter the Rubisco Activase derived polypeptides are heat treated priorto performance of the assay. Purified components can be used as well ascells comprising the components for Rubisco activation.

In other embodiments, Rubisco Activase derived polypeptide activity isassayed in vivo. Plants which do not express wild type Rubisco Activase(e.g., deletion mutants) are made to express one or more RubiscoActivase derived polypeptides and can be analyzed for photosynthesisrates, biomass, growth rates and seed yield under heated growingconditions. The plants may be grown entirely under heated conditions orfor shorter periods of time under heated conditions. In one embodiment,photosynthesis rates are measured by analyzing the plants for CO₂fixation. In another embodiment, growth rates are measured by analyzingleaf area of the plants. In another embodiment, seed yield is analyzedby determining seed weight from mature dried plants. In anotherembodiment, seed yield is analyzed by determining the seed germinationrate.

Methods of Enhancing Heat Tolerance in Plants

Rubisco Activase activates Rubisco in plants. Activated Rubisco isinvolved in photosynthesis and is the rate limiting step of thephotosynthetic process. With decreased Rubisco Activase activity,Rubisco remains inactive and photosynthesis slows or stops. Increasedtemperatures destabilize and/or denature Rubisco Activase therebyrendering the enzyme less able or unable to convert inactive Rubisco tothe active form. The present invention is directed to Rubisco Activasederived polypeptides with improved thermostability as compared to wildtype Rubisco Activase. As such, the photosynthetic process can be mademore heat tolerant with the involvement of Rubisco Activase derivedpolypeptides with improved thermostability.

Any method known in the art can be used to cause plants to express oneor more of the Rubisco Activase derived polypeptides of the invention.In one embodiment, transgenic plants can be made to express one or morepolypeptides of the invention. The transgenic plant may express the oneor more polypeptides of the invention in all tissues (e.g., globalexpression). Alternatively, the one or more polypeptides of theinvention may be expressed in only a subset of tissues (e.g., tissuespecific expression), preferably those tissues or organelles involved inphotosynthesis (e.g., the plastids). Polypeptides of the invention canbe expressed constitutively in the plant or be under the control of aninducible promoter. In some embodiments, the expression and/or activityof the endogenous Rubisco Activase of the plant is reduced oreliminated.

Recombinant Expression

Nucleic acid molecules and polypeptides of the invention can beexpressed recombinantly using standard recombinant DNA and molecularcloning techniques that are well known in the art (e.g., Sambrook,Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual; ColdSpring Harbor Laboratory Press: Cold Spring Harbor, (1989)).Additionally, recombinant DNA techniques may be used to create nucleicacid constructs suitable for use in making transgenic plants.

Accordingly, an aspect of the invention pertains to vectors, preferablyexpression vectors, comprising a nucleic acid molecule of the inventionor a variant thereof. As used herein, the term “vector” refers to apolynucleotide capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe introduced. Another type of vector is a viral vector, whereinadditional DNA segments can be introduced into the viral genome.

Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal vectors). Other vectors(e.g., non-episomal vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses).

The recombinant expression vectors of the invention comprise a nucleicacid molecule of the invention in a form suitable for expression of thenucleic acid molecule in a host cell. This means that the recombinantexpression vectors include one or more regulatory sequences, selected onthe basis of the host cells to be used for expression, which is operablyassociated with the polynucleotide to be expressed. Within a recombinantexpression vector, “operably associated” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described in the art (e.g., Goeddel, GeneExpression Technology: Methods in Enzymology, (1990) Academic Press, SanDiego, Calif.). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, thearea of the organism in which expression is desired, etc. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids molecules as described herein.

In some embodiments, isolated nucleic acids which serve as promoter orenhancer elements can be introduced in the appropriate position(generally upstream) of a non-heterologous form of a polynucleotide ofthe present invention so as to up or down regulate expression of apolynucleotide of the present invention. For example, endogenouspromoters can be altered in vivo by mutation, deletion and/orsubstitution (see, U.S. Pat. No. 5,565,350; International PatentApplication Number PCT/US93/03868) or isolated promoters can beintroduced into a plant cell in the proper orientation and distance froma cognate gene of a polynucleotide of the present invention so as tocontrol the expression of the gene. Gene expression can be modulatedunder conditions suitable for plant growth so as to alter the totalconcentration and/or alter the composition of the polypeptides of thepresent invention in plant cell. Thus, the present invention providescompositions, and methods for making heterologous promoters and/orenhancers operably linked to a native, endogenous (i.e.,non-heterologous) form of a polynucleotide of the present invention.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene or less preferably from any other eukaryotic gene.

The recombinant expression vectors of the invention can be designed forexpression of a polypeptide of the invention in prokaryotic (e.g.,Enterobacteriaceae, such as Escherichia; Bacillaceae; Rhizoboceae, suchas Rhizobium and Rhizobacter; Spirillaceae, such as photobacterium;Zymomonas; Serratia; Aeromonas; Vibrio; Desulfovibrio; Spirillum;Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter,Azotobacteraceae and Nitrobacteraceae) or eukaryotic cells (e.g., insectcells using baculovirus expression vectors, yeast cells, plant cells ormammalian cells) (see, Goeddel, supra. for a discussion on suitable hostcells). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors comprising constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve at least three purposes: 1) to increaseexpression of the recombinant protein; 2) to increase the solubility ofthe recombinant protein and/or 3) to aid in the purification of therecombinant protein by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantprotein to enable separation of the recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase. Typical fusion expression vectors include pGEX (PharmaciaBiotech Inc; Smith and Johnson, (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein orprotein A, respectively, to the target recombinant protein.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz, etal., (1987) Gene 54:113-123), pYES2 (Invitrogen Corp., San Diego,Calif.), and pPicZ (Invitrogen Corp., San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith, et al.,(1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow andSummers, (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid molecule of the invention isexpressed in plant cells using a plant expression vector including, butnot limited to, tobacco mosaic virus and potato virus expressionvectors.

Other suitable expression systems for both prokaryotic and eukaryoticcells are known in the art (see, e.g., chapters 16 and 17 of Sambrook,et al., (1990) Molecular Cloning, A Laboratory Manual, 2d Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-specific, inducible orother promoters for expression in the host organism.

A “tissue-specific promoter” may direct expression of nucleic acids ofthe present invention in a specific tissue, organ or cell type.Tissue-specific promoters can be inducible. Similarly, tissue-specificpromoters may only promote transcription within a certain time frame ordevelopmental stage within that tissue. Other tissue specific promotersmay be active throughout the life cycle of a particular tissue. One ofordinary skill in the art will recognize that a tissue-specific promotermay drive expression of operably linked sequences in tissues other thanthe target tissue. Thus, as used herein, a tissue-specific promoter isone that drives expression preferentially in the target tissue or celltype, but may also lead to some expression in other tissues as well. Anumber of tissue-specific promoters can be used in the presentinvention. With the appropriate promoter, any organ can be targeted,such as shoot vegetative organs/structures (e.g., leaves, stems andtubers), roots, flowers and floral organs/structures (e.g., bracts,sepals, petals, stamens, carpels, anthers and ovules), seed (includingembryo, endosperm and seed coat) and fruit. For instance, promoters thatdirect expression of nucleic acid molecules in leaves and/orphotosynthetic organ-specific promoters (such as the RBCS promoterdisclosed in Khoudi, et al., (1997) Gene 197:343) are useful forenhancing photosynthesis. Additionally, tissue specific expression canbe obtained by adding a peptide onto a polypeptide of the invention thatdirects localization of the attached polypeptide to the photosyntheticorgans (such as those disclosed in U.S. patent application Ser. No.11/150,054).

A “constitutive promoter” is defined as a promoter which will directexpression of a gene in all tissues and are active under mostenvironmental conditions and states of development or celldifferentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens, andother transcription initiation regions from various plant genes known tothose of ordinary skill in the art. Such genes include for example,ACT11 from Arabidopsis (Huang, et al., (1996) Plant Mol. Biol.33:125-139), Cat3 from Arabidopsis (GenBank Accession Number U43147,Zhong, et al., (1996) Mol. Gen. Genet. 251:196-203), the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankAccession Number X74782, Solocombe, et al., (1994) Plant Physiol.104:1167-1176), GPc1 from maize (GenBank Accession Number X15596,Martinez, et al., (1989) J. Mol. Biol. 208:551-565), and Gpc2 from maize(GenBank Accession Number U45855, Manjunath, et al., (1997) Plant Mol.Biol. 33:97-112). Any strong, constitutive promoter, such as the CaMV35S promoter, can be used for the expression of polynucleotides of thepresent invention throughout the plant.

The term “inducible promoter” refers to a promoter that is under preciseenvironmental or developmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions, elevated temperature, the presence of light orspraying with chemicals/hormones.

Suitable constitutive promoters for use in a plant host cell include,for example, the core promoter of the Rsyn7 promoter and other relatedconstitutive promoters (International Publication Number WO 99/43838 andU.S. Pat. No. 6,072,050); the core CaMV 35S promoter (Odell, et al.,(1985) Nature 313:810-812); rice actin (McElroy, et al., (1990) PlantCell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant Mol. Biol.12:619-632 and Christensen, et al., (1992) Plant Mol. Biol. 18:675-689);pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten,et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No.5,659,026) and the like (e.g., U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and6,177,611).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

Accordingly, the present invention provides a host cell having anexpression vector comprising a nucleic acid molecule of the invention,or a variant thereof. A host cell can be any prokaryotic (e.g., E. coli,Bacillus thuringiensis) or eukaryotic cell (e.g., insect cells, yeast orplant cells). The invention also provides a method for expressing anucleic acid molecule of the invention thus making the encodedpolypeptide comprising the steps of i) culturing a cell comprising anucleic acid molecule of the invention under conditions that allowproduction of the encoded polypeptide; and ii) isolating the expressedpolypeptide.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid molecules into a host cell, including calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, electroporation, Agrobacterium tumefaciens and vacuuminfiltration. Suitable methods for transforming or transfecting hostcells can be found in the art (e.g., Sambrook, et al., supra.).

Production of Transgenic Plants

Any method known in the art can be used for transforming a plant orplant cell with a nucleic acid molecule of the present invention.Nucleic acid molecules can be incorporated into plant DNA (e.g., genomicDNA or chloroplast DNA) or be maintained without insertion into theplant DNA (e.g., through the use of artificial chromosomes). Suitablemethods of introducing nucleic acid molecules into plant cells includemicroinjection (Crossway, et al., (1986) Biotechniques 4:320-334);electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci.83:5602-5606; D'Halluin, et al., (1992) Plant Cell 4:1495-1505);Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840, Osjoda, et al., (1996) Nature Biotechnology 14:745-750;Horsch, et al., (1984) Science 233:496-498; Fraley, et al., (1983) Proc.Natl. Acad. Sci. 80:4803; and Gene Transfer to Plants, Potrykus, ed.,Springer-Verlag, Berlin 1995); direct gene transfer (Paszkowski, et al.,(1984) EMBO J. 3:2717-2722); ballistic particle acceleration (U.S. Pat.Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes, et al., (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment, in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips, Springer-Verlag, Berlin; and McCabe,et al., (1988) Biotechnology 6:923-926); virus-mediated transformation(U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and5,316,931); pollen transformation (De Wet, et al., (1985) in TheExperimental Manipulation of Ovule Tissues, ed. Chapman, et al.,Longman, N.Y., pp. 197-209); Lec 1 transformation (U.S. patentapplication Ser. No. 09/435,054; International Patent Publication NumberWO 00/28058); whisker-mediated transformation (Kaeppler, et al., (1990)Plant Cell Reports 9:415-418; Kaeppler, et al., (1992) Theor. Appl.Genet. 84:560-566) and chloroplast transformation technology (Bogorad,(2000) Trends in Biotechnology 18:257-263; Ramesh, et al., (2004)Methods Mol. Biol. 274:301-7; Hou, et al., (2003) Transgenic Res.12:111-4; Kindle, et al., (1991) Proc. Natl. Acad. Sci. 88:1721-5;Bateman and Purton, (2000) Mol Gen Genet. 263:404-10; Sidorov, et al.,(1999) Plant J. 19:209-216).

The choice of transformation protocols used for generating transgenicplants and plant cells can vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Examples oftransformation protocols particularly suited for a particular plant typeinclude those for: potato (Tu, et al., (1998) Plant Molecular Biology37:829-838; Chong, et al., (2000) Transgenic Research 9:71-78); soybean(Christou, et al., (1988) Plant Physiol. 87:671-674; McCabe, et al.,(1988) BioTechnology 6:923-926; Finer and McMullen, (1991) In Vitro CellDev. Biol. 27P:175-182; Singh, et al., (1998) Theor. Appl. Genet.96:319-324); maize (Klein, et al., (1988) Proc. Natl. Acad. Sci.85:4305-4309; Klein, et al., (1988) Biotechnology 6:559-563; Klein, etal., (1988) Plant Physiol. 91:440-444; Fromm, et al., (1990)Biotechnology 8:833-839; Tomes, et al., (1995) “Direct DNA Transfer intoIntact Plant Cells via Microprojectile Bombardment,” in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg(Springer-Verlag, Berlin)); cereals (Hooykaas-Van Slogteren, et al.,(1984) Nature 311:763-764; U.S. Pat. No. 5,736,369).

In some embodiments, more than one construct is used for transformationin the generation of transgenic plants and plant cells. Multipleconstructs may be included in cis or trans positions. In preferredembodiments, each construct has a promoter and other regulatorysequences.

The transgenic plants can express the transgene in any way known in theart including, but not limited to, constitutive expression,developmentally regulated expression, and tissue specific expression. Ina specific embodiment, promoters that direct expression of nucleic acidmolecules in leaves and/or photosynthetic organs (such as the RBCSpromoter disclosed in Khoudi, et al., Gene 197:343) are used to expressthe nucleic acid molecules and/or polypeptides of the invention.

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker that has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in the art (e.g., Evans, et al., ProtoplastsIsolation and Culture, Handbook of Plant Cell Culture, pp. 124-176,MacMillilan Publishing Company, New York, 1983; and Binding,Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, BocaRaton, 1985). Regeneration can also be obtained from plant callus,explants, organs or parts thereof. Such regeneration techniques are alsodescribed in the art (e.g., Klee, et al., (1987) Ann. Rev. of Plant Phys38:467-486).

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g. leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g. bracts, sepals, petals, stamens, carpels,anthers and ovules), seed (including embryo, endosperm and seed coat)and fruit (the mature ovary), plant tissue (e.g. vascular tissue, groundtissue and the like) and cells (e.g. guard cells, egg cells, trichomesand the like) and progeny of same. The class of plants that can be usedin methods of the present invention includes the class of higher andlower plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), gymnosperms,ferns and multicellular algae. Plants of a variety of ploidy levels,including aneuploid, polyploid, diploid, haploid and hemizygous plantsare also included.

The nucleic acid molecules of the invention can be used to conferdesired traits on essentially any plant. Thus, the invention has useover a broad range of plants, including species from the genera Agrotis,Allium, Ananas, Anacardium, Apium, Arachis, Asparagus, Athamantha,Atropa, Avena, Bambusa, Beta, Brassica, Bromus, Browaalia, Camellia,Cannabis, Carica, Ceratonia. Cicer, Chenopodium, Chicorium, Citrus,Citrullus, Capsicum, Carthamus, Cocos, Coffea, Coix, Cucumis, Cucurbita,Cynodon, Dactylis, Datura, Daucus, Dianthus, Digitalis, Dioscorea,Elaeis, Eliusine, Euphorbia, Festuca, Ficus, Fragaria, Geranium,Glycine, Graminae, Gossypium, Helianthus, Heterocallis, Hevea, Hibiscus,Hordeum, Hyoscyamus, Ipomoea, Lactuca, Lathyrus, Lens, Lilium, Linum,Lolium, Lotus, Lupinus, Lycopersicon, Macadamia, Macrophylla, Malus,Mangifera, Manihot, Majorana, Medicago, Musa, Narcissus, Nemesia,Nicotiana, Onobrychis, Olea, Olyreae, Oryza, Panicum, Panicum, Panieum,Pannisetum, Pennisetum, Petunia, Pelargonium, Persea, Pharoideae,Phaseolus, Phleum, Picea, Poa, Pinus, Pistachia, Pisum, Populus,Pseudotsuga, Pyrus, Prunus, Pseutotsuga, Psidium, Quercus, Ranunculus,Raphanus, Ribes, Ricinus, Rhododendron, Rosa, Saccharum, Salpiglossis,Secale, Senecio, Setaria, Sequoia, Sinapis, Solanum, Sorghum,Stenotaphrum, Theobromus, Trigonella, Trifolium, Trigonella, Triticum,Tsuga, Tulipa, Vicia, Vitis, Vigna and Zea.

In specific embodiments, transgenic plants are maize, tomato, potato,rice, soybean, cotton, sunflower, alfalfa, lettuce, canola, sorghum ortobacco plants.

Transgenic plants may be grown and pollinated with either the sametransformed strain or different strains. Two or more generations of theplants may be grown to ensure that expression of the desired nucleicacid molecule, polypeptide and/or phenotypic characteristic is stablymaintained and inherited. One of ordinary skill in the art willrecognize that after the nucleic acid molecule of the present inventionis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

Determination of Expression in Transgenic Plants

Any method known in the art can be used for determining the level ofexpression in a plant of a nucleic acid molecule of the invention orpolypeptide encoded therefrom. For example, the expression level in aplant of a polypeptide encoded by a nucleic acid molecule of theinvention can be determined using molecular techniques including, butnot limited to, immunoassay, immunoprecipitation, gel electrophoresisand quantitative gel electrophoresis.

Additionally, the expression level in a plant of a polypeptide encodedby a nucleic acid molecule of the invention can be determined by thedegree to which the plant phenotype (including, but not limited to,photosynthesis rates, growth rates, and seed yield) is altered underheated conditions compared to plants expressing wild type RubiscoActivase.

Furthermore, extracts or polypeptides isolated from transgenic plants,tissues thereof, or cells thereof can be used in in vitro assays.

The contents of all published articles, books, reference manuals andabstracts cited herein, are hereby incorporated by reference in theirentirety to more fully describe the state of the art to which theinvention pertains.

As various changes can be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above descriptionand/or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Modifications and variations ofthe present invention are possible in light of the above teachings.

EXAMPLES Example 1 Isolating Rubisco Activase Derived Polypeptides

Rubisco Activase libraries were generated from single gene shuffling(see, e.g., Crameri, et al., (1998) Nature. 391(6664):288-91; Chang, etal., (1999) Nat. Biotechnol. 17(8):793-7; Ness, et al., (1999) Nat.Biotechnol. 17(9):893-6; Christians, et al., (1999) Nat. Biotechnol.17(3):259-64 and U.S. Pat. Nos. 6,605,430; 6,117,679 and 5,605,793) andsynthetic shuffling (see, e.g., U.S. Pat. No. 6,436,675 andInternational Publication Numbers WO 00/42561; WO 01/23401; WO 00/42560;and WO 00/42559) using wild type Rubisco Activase of SEQ ID NO: 1 as atemplate.

Briefly, Arabidopsis RNA was isolated from green leaves using Trizol®reagent according to the manufacturer's protocol (Invitrogen). RCA cDNA(GenBank accession number NM 179990) was PCR cloned into TOPO® vector(Invitrogen) using TITANIUM™ one-step RT-PCR Kit (BDBiosciences-Clontech). For single gene shuffling in the first round, themature RCA short form (coding region V59 to K438) was PCR amplified(Qiagen Taq DNA polymerase or Stratagene Mutazyme DNA polymerase),fragmented and reassembled in a primerless PCR reaction and the shuffledgenes were then rescued with flanking primers that contain an NcoI site(5′) and a BamHI site, 6×-His coding region and a stop codon (3′). Thelibrary of variants was cloned into an E. coli expression vector(pET16b, Novagen) digested with NcoI and BamHI. To increase pool ofgenetic in the first round, synthetic shuffling was carried out usingdiversity from wheat, rice, cotton, spinach and cucumber (see, Ness, etal., (2002) Nat. Biotechnol. 20:1251-1255). A second round of geneshuffling using first round variants as parents was performed aspreviously described by Crameri, et al., (1998) Nature 15:288-291.

Rubisco Activase derived polypeptides with improved thermostability wereisolated by the following screening methodology.

First tier: Rubisco Activation Assay. Cultures of E. coli expressing aRubisco Activase derived polypeptide were pelleted at 4° C., 3,500 rpmfor 15 minutes and stored in a 96-well V-bottom PCR-plate at −80° C. E.coli cell lysate was prepared by thawing the cultures at roomtemperature for 5 minutes, adding 75 μl Sonication buffer (100 mMTricine KOH pH 8.0; 20 mM ascorbate; 3 mM Mg-ATP; 10 mM MgCl₂; 10% v/vglycerol; 10 mM βme; x3.33 protease inhibitor; 1 μl/ml benzonase; 1mg/ml lysosyme) to each well and shaking the plate for 60 minutes at 4°C. until the pellet was lysed. The plates were sonicated with MISONIXmicroplate sonicator for 1 minute and then cooled for 1 minute. Thisprocess was repeated four times. The cultures were centrifuged at 4,000rpm for 20 minutes at 4° C. The supernatant that contained solubleprotein was used in the Rubisco activation assays.

Twenty-two μl of the E. coli supernatant was transferred to a 96-wellU-bottom scintillation-plate and incubated at room temperature for 15minutes (and used as the lysate for “normal conditions” in the assays).Heat treatment of the lysate was performed by transferring 35 μl of theE. coli supernatant to a 96-well V-bottom PCR-plate and incubating at40° C. for 15 minutes. The heat treated supernatant was then transferredto a 96-well U-bottom scintillation-plate (and used as the lysate for“heat treated conditions” in the assays). Both plates were incubated for5 minutes at 4° C. before assay performance.

Rubisco activation was assayed by incubating the cell lysate containingRubisco Activase or a Rubisco Activase derived polypeptide with purifieddeactivated Arabidopsis Rubisco (15 μg) in reaction buffer (100 mMTricine KOH pH 8.0; 10 mM MgCl₂; 10 mM [¹⁴C]NaHCO₃; Mg-ATP; 4 mM RuBP; 1mM PEP; 40 μg/ml pyruvate kinase) at room temperature for 15 minutes(see, Shen, et al., (1991) J. Biol. Chem. 266:8963-8968). The activationof Rubisco by cell lysate expressing Rubisco Activase derivedpolypeptides was terminated by addition of 1 N HCl, and theincorporation of ¹⁴CO₂ determined by liquid scintillation spectroscopy.

The Rubisco used in the above-described assay was purified fromArabidopsis leaves. The leaves were homogenized and frozen in liquidnitrogen before resuspension in extraction buffer (100 mM Hepes-KOH pH8.0; 1 mM EDTA pH 8.0; 3 mM DDT; 0.5 mM PMSF; 10 mM MgCl₂; 10 mMNaHCO₃). The suspension was centrifuged at 12,000 rpm for 20 min at 4°C. and the supernatant was collected. The supernatant was kept on iceunder continuous stirring while ammonium sulfate was added to a finalconcentration of 35% of saturation and centrifuged at 12,000 rpm for 20min at 4° C. The supernatant was stirred for an additional 30 min. withammonium sulfate to a final concentration of 55% of saturation beforecentrifugation at 12,000 rpm for 20 min at 4° C. The pellet wasdissolved in extraction buffer and further precipitated with 18%polyethylene glycol and centrifugation. The pellet was resuspended inextraction buffer (about 1 ml/original 40 ml of supernatant) andcentrifuged at 13,000 rpm for 30 min at 4° C. The purified Rubisco wasin the supernatant and glycerol was added to a final concentration of10%.

The purified Rubisco was deactivated by the following protocol (see,Wang, et al., (1992) Plant Physiol. 100:1858-1862). Ten mM of DTT wasadded to the purified Rubisco and incubated at 45° C. for 10 min. One mlof the mixture was added to a 20 ml Sephadex G-50 column equilibratedwith equilibration buffer (50 mM Tricine-KOH pH 8.0 and 0.5 mM EDTA pH8.0). Deactivated Rubisco was eluted from the column by the addition of1 ml fractions of equilibration buffer. The eluted deactivated Rubiscowas collected and incubated at room temperature to 1 hour beforeincubation on ice for one hour in the presence of 4 mM RuBp.

Second tier: HTP temperature profile of Rubisco activation by celllysate of active clones. Clones of interest identified in the first tierscreening were further characterized in the second tier of screening.Cell lysate from each active clone was retested as described aboveexcept that the temperature treatment was carried out at four differenttemperatures (16° C., 25° C., 40° C. and 45° C.) prior to assay. Clonesthat possessed relatively improved thermostability profile compare towide type Rubisco Activase (SEQ ID NO: 2) were selected for 3^(rd) tierscreening.

Third tier: Temperature profile of Rubisco activation by purifiedRubisco Activase variants. In order to determine the specific activityof the derived polypeptides identified by the first two tiers ofscreening, affinity purified polypeptides were pre-incubated atdifferent temperatures and then analyzed for their ability to activateRubisco at 25° C. Since Rubisco Activase catalyses deactivated Rubiscoin a time dependent manner, each reaction was monitored for 15 minutesin 3-minute intervals. The ratio of Rubisco Activase:Rubisco was set to1:40 similarly to the ratio in plant leaves. The thermostability of wildtype Rubisco Activase (SEQ ID NO: 2) and Rubisco Activase derivedpolypeptides is shown in Table 2. The percent thermostability representsthe amount of Rubisco that has been activated by Rubisco Activase at 40°C. as a percent of the amount of Rubisco that has been activated byRubisco Activase at 25° C.

Example 2 In vitro characterization of Rubisco Activase DerivedPolypeptides

The Rubisco Activase derived polypeptides isolated in Example 6.1 weretested in vitro in three different assays in order to determine thespecific activity at 25° C. and 40° C. and their thermostability (t.s.).In all cases, the results obtained with wild type Rubisco Activase at25° C. were set to 100%.

Activation of deactivated Rubisco. Purified Rubisco Activase derivedpolypeptides were assayed as described in the first tier assay ofExample 1 except that the derived polypeptides were incubated at 40° C.or 45° C. for 15, 30, 45 or 60 minutes prior to performance of theRubisco activation assay. Results are shown in columns 2-4 of Table 3.Column 2 of Table 3 represents the amount of activated Rubisco that isobtained after incubation of deactivated Rubisco with Rubisco Activaseat 25° C. There were no heated conditions used. Column 3 of Table 3represents the amount of Rubisco that has been activated by RubiscoActivase after a 15 min 40° C. heat treatment as a percent of the amountof Rubisco that has been activated by Rubisco Activase at 25° C. Column4 of Table 3 represents the amount of Rubisco that has been activated byRubisco Activase with a 45 min 40° C. heat treatment as a percent of theamount of Rubisco that has been activated by Rubisco Activase with noheat treatment (at 25° C.).

Rubisco activation by Rubisco Activase derived polypeptides 301C7 and382D8 exhibit high thermostability at 40° C. and 45° C. treatments (FIG.1A). The activity of 382D8 after 45° C. treatment was 80% higher thanRCA1 at the same temperature treatment and only 10% less than theactivity of RCA1 incubated at 25° C.

Rubisco activation under catalytic conditions. Purified Rubisco Activasederived polypeptides were assayed as described in the first tier assayof Example 1 except that the assay was performed under heated conditions(i.e., 40° C.) (Crafts-Brandner and Salvucci, (2000) PNAS97:13430-13435). Results are shown in columns 5-6 of Table 3. Column 5of Table 3 represents the amount of activated Rubisco that is obtainedafter incubation of deactivated Rubisco with Rubisco Activase at 25° C.There were no heated conditions used. Column 6 of Table 3 represents theamount of Rubisco that has been activated by Rubisco Activase when theassay is conducted at 40° C. as a percent of the amount of Rubisco thathas been activated by Rubisco Activase when the assay is conducted at25° C.

Wild-type RCA maintained a Rubisco activation state of 0.5 at 40° C.while Rubisco Activase derived polypeptides 183H12, 301C7 and 382D8 wereable to maintain activation states of 0.62-0.72 under the sameconditions (FIG. 1B). Relative to reactions at 25° C., the activationstate of Rubisco maintained by the thermostable variants at 40° C. wasin the range of 78-98% versus 70% for the wild type enzyme. The proteindisplaying the highest specific activity at either 25° C. or 40° C. wasthe best variant isolated in the first round, 183H12.

ATPase activity. Since Rubisco Activase is an ATPase (the polypeptidecontains the AAA⁺ domain) that requires ATP to loosen the binding ofRubisco to sugar phosphates, the effect of temperature on ATP hydrolysisby Rubisco Activase was tested. The ATPase assay that monitored theintrinsic activity of the activase complex regardless of its interactionwith Rubisco is commonly used for Rubisco Activase characterization.ATPase assay has been performed as described by Salvucci ((1992) Arch.Biochem. Biophys. 298:688-696). Results are shown in columns 7-8 ofTable 3. Column 7 of Table 3 represents the amount of hydrolyzed ATPthat is present after incubation of Rubisco Activase with ATP at 25° C.There were no heated conditions used. Column 8 of Table 3 represents theamount of hydrolyzed ATP that is present after incubation with RubiscoActivase that had been heat treated at 40° C. as a percent of the amountof hydrolyzed ATP that is present after incubation with Rubisco Activasethat had not been heat treated.

FIG. 1C shows that the stability of Rubisco Activase derivedpolypeptides 301C7 and 382D8 at 35° C. and 40° C. was improved more than10-fold compared to RCA1, whereas 183H12 exhibited 20% and 30%improvement at 25° C. and 40° C., respectively.

Example 3 Complementation of Rubisco Activase Deletion Mutant

In order to express shuffled variants in homozygous background for thedeletion (Δrca/Δrca) (see, Li, et al., (2001) Plant J. 27:235-242) thefollowing complementation cascade was developed: 1) Selection ofheterozygous plants for the deletion by HTP-PCR using single-leaf96-well DNA extraction method (Xin, et al., (2003) BioTechniques34:820-826), with specific primers for the wild-type and deletedalleles. 2) Transformation with the gene of interest. 3) TO selectionfor antibiotic resistance and PCR analysis for homozygosity. 4) Selfpollination of the resultant homozygous plants in order to obtain T1transgenic lines.

Immunoblot analysis of wild-type, heterozygous, and homozygous plants(genetic background RCA/RCA, RCA/Δrca and Δrca/Δrca respectively)revealed that the gene products (long and short forms) were expressed atsimilar levels in wild-type and heterozygous plants (FIG. 2A). Theabsence of the short and long isoforms in plants homozygous for thedeletion confirmed that the mutation abrogates the expression of bothRCA1 and RCA2. Δrca plants grown at ambient CO₂ exhibited lowphotosynthetic performance (Fq′/Fm′ values) compared to wild-type(0.185±0.038 and 0.332±0.033 respectively) (FIG. 2B) and significantlower leaf area after 3 weeks on soil (2.93±0.49 and 395.4±8.75 mm²respectively) (FIG. 2C). Two-month old Δrca homozygotes were severelystunted and chlorotic by comparison to wild-type plants (FIG. 2D).

Based on sequence analysis and mapping of the deleted fragment, two setsof primers were designed: RCA primers (forward 5′-CAGACAATGTTGGCCTC-3′(SEQ ID NO: 23) and reverse 5′-ACGAGTAACGATGGTAGG-3′ (SEQ ID NO: 24))specific for the wild-type allele that give 1.5 kb product, and rcaprimers (forward 5′-GTCTATACCTTGAGC-3′ (SEQ ID NO: 25) and reverse5′-TCAGTCATACTCGG-3′ (SEQ ID NO: 26)) that give 1.5 kb product in thedeleted allele and 4.9 kb in the wild-type allele (FIG. 3A). In order toamplify the 1.5 kb product with the rca primers but not the 4.9 kb, thePCR amplification cycle was set to 1.5 min. Those two sets of primerswere utilized to characterize the genetic background of the T1 plants.Since the transformation host was heterozygous for deletion of theendogenous rca locus, the T1 plants expressing the Rubisco activasetransgenes are a mixture of wild-type (RCA/RCA) heterozygotes (RCA/Δrca)and homozygotes (Δrca/Δrca). Transgenic lines expressing the shuffledvariant 183H12 in the different genetic backgrounds have been identifiedusing PCR screening (FIG. 3B) and immunoblot analysis (FIG. 3C). Plantsthat express 183H12 in genetic backgrounds containing at least one wildtype allele (#13; wt, #12; heterozygous for the deletion) have both theshort and the long forms of the protein. In line #2 (homozygous for thedeletion) only the short form was detected because the transgene wasdesigned to express only the short form of the protein.

Example 4 In Planta Characterization of Rubisco Activase DerivedPolypeptides

To determine the effect of improved Rubisco Activase under normal andincreased temperatures, the Arabidopsis Rubisco Activase deletion mutant(Δrca) (see, Example 3). Δrca was functionally complemented with widetype Rubisco Activase (SEQ ID NO: 1), 1^(st) round Rubisco Activasederived polypeptide 183H12 (SEQ ID NO: 7) and two 2^(nd) round RubiscoActivase derived polypeptides 382D8 (SEQ ID NO: 15) and 301C7 (SEQ IDNO: 19).

In order to express the wild type Rubisco Activase and the RubiscoActivase derived polypeptides in transgenic Arabidopsis plants (Δrca),the transgenes encoding the chloroplast transit peptide and the codingregion of rcal or the derived polypeptides were cloned into pMAXY4384that contains the Mirabilis Mosaic Caulimovirus promoter (MMV) with adouble enhancer domain (Day and Maiti, (1999) Transgenics 3:61-70), theUBQ3 terminator and the kanamycin resistance gene nptII. HeterozygousDeleteagene™ RCA mutants were transformed by Agrobacterium tumefaciensstrain GV3101 using the floral dipping method (Clough, et al., (1998)Plant J. 16:735-743). To confirm expression, protein was extracted fromplant tissue (2-3 g fresh weight) in liquid N₂ and 1 ml of extractionbuffer (100 mM Tricine-KOH pH 8, EDTA pH 8, 10 mM 2-mercaptoethanol, andProtease inhibitor cocktail Set V). The crude extract was clarified bysuccessive centrifugation for 5 min at 3000 g, and 20 min at 12,000 g.Ten micrograms of soluble protein extract was separated on 10%SDS-polyacrylamide gels and transferred to a nitrocellulose membrane(according to the instructions supplied by Invitrogen). The blot wasimmunodecorated with the polyclonal antibodies raised against therecombinant Arabidopsis RCA1 and the proteins were detected using the Apconjugated substrate kit (Bio-Rad).

As shown in FIG. 4A, wild-type plants (RCA/RCA) expressed short and longisoforms of activase, whereas transgenic Δrca lines complemented by thetransgenes expressed only the 43 kDa short isoform. Under 22° C. cultureconditions (plants grown in 16 hour light (225 μmol photons m⁻² s⁻¹)/8hour dark cycles), the transgenic lines exhibited similar growth ratesas the wild-type untransformed plants (FIG. 4B). Photosyntheticperformance (photosystem II operating efficiency Fq′/Fm′) and growthrates were analyzed using the chlorophyll a fluorescence imaging system(Fluorlmager, Qubit Systems) as previously described (Baker, et al.,(2001) J. Exp. Bot. 52:615-621). The Fq′/Fm′ values of transgenicdeletion lines expressing RCA1 or Rubisco Activase derived polypeptides183H12, 301C7 or 382D8 (ΔrcaRCA1, Δrca183H12, Δrca301C7 and Δrca382D8,respectively) were similar to wild-type untransformed plants, indicatingthat expression of the short form is sufficient for functionalcomplementation of Δrca under normal growth conditions (FIG. 4C). Underthese conditions the photosynthetic activity measured by the portableinfrared gas analyzer (L16400, Li-Cor) under 150 μmol photons m⁻² s⁻¹and 350 pbar CO₂. of ΔrcaRCA1-1 was similar to Δrca183H12-3, Δrca301C7-3and Δrca382D8-1 (FIG. 4D). Temporary exposure to 30° C. for 1 hrresulted in 12% decreased photosynthesis in ΔrcaRCA1-1. Conversely,lines Δrcal 83H12-3, Δrca301C7-3 and Δrca382D8-1 showed 16, 22 and 16%increased photosynthesis after 1 hr at 30° C.

Since exposure of Arabidopsis plants to 30° C. causes minor induction ofheat shock proteins (typically induced at 32° C. and above) and minoreffects on stomatal aperture (Salvucci, et al., (2001) Plant Physiol.127:1053-1064), growth under prolonged heat treatment was conducted withArabidopsis plants expressing wild type or thermostable RubiscoActivase. Four-week old transgenic lines exposed for two weeks tomoderate heat stress. Conditions of growth were 16 hours of light at 225μmol photons m⁻² s⁻¹ and 8 hours of dark. During the light cycle, plantswere grown at 22° C. for 6 hours then rapidly increased to 30° C. (2° C.per min) for 4 hours and then returned to 22° C. for the completion ofthe light cycle. During the dark cycle, plants remained at 22° C.Characterization of growth, biomass, and yield was performed aspreviously described (Barth, et al., (2003) Heredity. 91:36-42). Theplants displayed normal phenotype and leaf color but varied in size(FIG. 5A). ΔrcaRCA1 (lines 1, 8 and 9) were stunted by comparison towild-type untransformed plants and to the Δrca lines that express theRubisco Activase derived polypeptides (FIG. 5B). Transgenic Arabidopsisexpressing 183H12-3 that possesses the highest in vitro specificactivity were the largest plants. Lines that expressed 301C7 and 382D8were larger than ΔrcaRCA1 lines but did not reach the leaf area levelsof the 183H12 lines. While Δrca lines expressing only the short form ofthe wild-type gene (RCA1) were smaller than wild-type untransformedlines (expressing both short and long forms), most transgenic linesexpressing the Rubisco Activase derived polypeptides exhibited greaterleaf area than wild-type untransformed plants (FIG. 5B), and all linesexpressing the Rubisco Activase derived polypeptides were significantlylarger (P=0.01) than the Δrca transformants expressing RCA1.

Four-week old plants exposed to two weeks of moderate heat stress alsoshowed differences in rates of plant development. At the end of thetreatment period 74, 44 and 33% of Δrca183H12, Δrca301C7 and Δrca382D8,respectively, had mature inflorescence with open flowers, while 100% ofuntransformed wild-type plants and 88% of ΔrcaRCA1 lines had emergingimmature inflorescences with no open flowers (data not shown).Additionally, 12% of the ΔrcaRCA1 lines were in the vegetative stagewith no visible inflorescences. Under normal growth conditions,Arabidopsis plants flower after four weeks. Therefore, the relativelyhigh percentage of Δrca183H12, Δrca301C7 and Δrca382D8 lines showingnormal development is likely due to improved thermostability of RCA thatminimized the inhibition of photosynthesis and growth under moderateheat stress conditions.

The best line from each variant was further analyzed for photosyntheticactivity during the moderate heat stress cycle (after 2 hr at 30° C.).Transgenic lines showed a CO₂ fixation pattern that correlated with leafarea. Rates of CO₂ fixation in lines Δrca183H12-3, Δrca301C7-3 andΔrca382D8-1 were 30, 25 and 23% higher, respectively, than in lineΔrcaRCA1-1. These results demonstrated that Rubisco activase is alimiting factor in photosynthesis under the experimental conditions.

Mature plants (10 weeks old) exposed for 8 weeks to moderate heat stresswere similar in appearance. A slight positive effect on plant height wasdetected in Δrca183H12-3, Δrca301C7-3 and Δrca382D8-1 lines (116, 121and 119% respectively) compared to ΔrcaRCA1-1 (FIG. 5D). A dramaticdifference was observed in the number of siliques per plant, which was130.8±48.2, 84.3±19.6 and 100.8±26.9 for Δrca183H12-3, Δrca301C7-3 andΔrca382D8-1 (respectively) compared to 40.2±16.3 and 47.5±15.8 forΔrcaRCA1-1 and wild-type, respectively (FIG. 5E).

To confirm that the relatively enhanced formation of siliques intransformants expressing improved RCA was not at the expense ofindividual seed size, the weights of lots of 1000 seeds were compared.As shown in FIG. 5F, Δrca183H12-3, and Δrca382D8-1 produced slightlylarger seeds (18 and 32% respectively) than ΔrcaRCA1-1, while the seedweight of Δrca301C7-3 and wild-type plants was similar to that ofΔrcaRCA1-1.

To further analyze the effect of improved RCA on growth under moderatetemperature stress, T3 lines expressing the most active clone at 25° C.(in vitro), 183H12, were grown continuously at 26° C. under higher lightintensity and humidity than in the previous experiment. Conditions ofgrowth were 16 hours of light at 300 μmol photons m⁻² s⁻¹ and 8 hours ofdark at 26° C. and 85% humidity. Wild-type and ΔrcaRCA1 lines grown at26° C. produced slightly decreased overall biomass and exhibited slowrates of plant development than under normal growth conditions, whereasthe biomass and the developmental process of lines of Δrca183H12 wasunchanged (not shown). In contrast, the number of siliques per plantproduced by Δrca plants grown at 26° C. was dramatically affected by theRubisco Activase derived polypeptide they expressed (FIG. 6A).Δrca183H12 lines possessed 50 to 100 more siliques per plant thanΔrcaRCA1 lines and 40 to 80 more siliques per plant than wild-typeplants. In addition, the siliques of Δrca183H12 were larger than thoseof wild-type plants and the ΔrcaRCA1 lines and produced more seeds (FIG.6B). Siliques from Δrca183H12 at 26° C. exhibited a similar phenotype tothe wild-type grown under normal growth conditions, but produced fewerseeds. Under normal growth conditions a minor decrease in seed weightwas observed in Δrca lines expressing RCA1 and 183H12 compared towild-type plants (FIG. 6C; white bars). However, under continuousexposure to 26° C., 50% to 150% greater seed weight was observed inlines of Δrca183H12 than in either wild-type plants or lines ofΔrcaRCA1. In comparing seed weight for each line grown at 26° C. to thatof the same line grown at 22° C., lines Δrca183H12-2, Δrca183H12-3 andΔrca183H12-20 were strikingly less affected by the higher growthtemperature than the ΔrcaRCA1 lines or the wild type.

Since exposure to 26° C. resulted in small siliques containing few seedsof small seed-weight, seed viability was analyzed using a germinationtest. Seeds from wild-type, ΔrcaRCA1-1 and Δrca183H12-3 were collectedfrom plants grown at normal growth conditions and 26° C. and thengerminated at 22° C. Seeds from ΔrcaRCA1-1 and Δrca183H12-3 lines ofparents grown at 22° C., showed the same germination rate (86%), whichwas slightly lower than that of wild-type plants (94%) (FIG. 6C).Complete inhibition of germination (4%) was observed in ΔrcaRCA1-1 seedscollected from parents grown at 26° C. and significant inhibition inwild-type seeds (26%). Conversely, Δrca183H12-3 seeds collected fromparents grown at 26° C. exhibited relatively high germination rates of70%.

Additionally plants were analyzed for photosynthesis rates, growth ratesand seed yield under the following growth conditions:

Normal: Plants were grown under 16 hours light (225 μmol photons m⁻²s⁻¹) and 8 hour dark regime at 22° C.

Increased temperatures: Plants were grown under normal growth conditionsfor two weeks and then transferred to the growth chamber and grown under16 hour light (225 μmol photons m⁻² s⁻¹) and 8 hour dark regime. Duringthe light cycle, the temperature was set to 22° C. for six hours andthen rapidly increased to 30° C. (2° C. per minute) for four hours.After the heat treatment, the temperature was set back to 22° C.

Continuous increased temperatures: Plants were grown under normal growthconditions for two weeks and then transferred to the growth room andgrown under sixteen hours high light (300 μmol photons m⁻² s⁻¹) andeight hour dark regime. During the light/dark cycle the temperature wasset to 26° C. and the humidity to 80%.

The results of the in planta assays of Rubisco Activase derivedpolypeptide activity at the conditions identified supra are summarizedbelow.

Growth rates. Leaf area was measured using the chlorophyll afluorescence imaging system (FluorImager, Qubit System Inc.). Leaf areaobserved in untransformed wild type plants under the different growthconditions was set to 100%. The data in Table 4 demonstrates that plantsexpressing any of the three Rubisco Activase derived polypeptides hadincreased growth rates under increased temperatures as compared toeither wild type plants or plants expressing a transgenic wild typeRubisco Activase.

Photosynthesis rates. Plants were analyzed for CO₂ fixation using theportable infrared gas analyzer (L16400, Li-Cor) for 15 minutes. Thelight source was set to 225 μmol photons m⁻² s⁻¹ and the level of CO₂supplied to the leaf by the built-in CO₂ injection system was 350 μmolm⁻² s⁻¹. The data in Table 5 shows that plants expressing any of thethree Rubisco Activase derived polypeptides had increased photosyntheticrates under increased temperatures as compared to either wild typeplants or plants expressing a transgenic wild type Rubisco Activase(see, column 3).

Seed yield. Seed weight (mg) was determined from mature dried plants.Seed germination rate was determined by number of plants germinated onMS plate supplemented with Kanamycin. The data in Table 5 shows thatplants expressing any of the three Rubisco Activase derived polypeptideshad increased seed yield under increased temperatures as compared toeither wild type plants or plants expressing a transgenic wild typeRubisco Activase (see, column 4). The increased seed yield was alsopresent in plants expressing any of the three Rubisco Activase derivedpolypeptides under continuous increased temperature conditions (see,Table 6). Germination rates of seeds was increased under continuousincreased temperature conditions in plants expressing the RubiscoActivase derived polypeptide 183H12 (SEQ ID NO: 7) as compared to eitherwild type plants or plants expressing a transgenic wild type RubiscoActivase (see, Table 7).

TABLE 1 Codon Table Amino acid Codon Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

TABLE 2 Effect of amino acids substitution on Rubisco Activase activityand thermostability. Activity (3^(rd) tier) Thermo- Amino acid positionμmol CO₂ min⁻¹ mg⁻¹ stability* Clone 42 130 131 168 257 274 293 310 25°C. 40° C. (%) Wild type M M M F V T R K  0.96 ± 0.04 0.634 ± 0.02 66(SEQ ID NO: 1) 126H4 R 0.616 ± 0.04 0.596 ± 0.05 97 (SEQ ID NO: 3)182B11 I 0.426 ± 0.03 0.359 ± 0.01 84 (SEQ ID NO: 5) 183H12 R 1.049 ±0.02 0.857 ± 0.01 82 (SEQ ID NO: 7) 184B2 N 1.055 ± 0.03 0.889 ± 0.02 84(SEQ ID NO: 9) 079H6 T I K 1.064 ± 0.01 0.940 ± 0.02 88 (SEQ ID NO: 11)214A4 T R 1.069 ± 0.06 0.986 ± 0.04 92 (SEQ ID NO: 13) 382D8 V I N 1.012± 0.05  0.91 ± 0.06 90 (SEQ ID NO: 15) 383A12 L I R N 0.891 ± 0.02 0.920± 0.02 103 (SEQ ID NO: 17) 301C7 L I N 0.905 ± 0.04 0.889 ± 0.01 98 (SEQID NO: 19) 301H3 I R N 0.971 ± 0.05 0.860 ± 0.05 89 (SEQ ID NO: 21)*Percent of activated Rubisco at 40° C. compared to amount of activatedRubisco 25° C.

TABLE 3 Relative activity (%) of Rubisco Activase Derived Polypeptides.Activation of deactivated Rubisco activation under Rubisco assaycatalytic conditions assay ATPase assay T.S T.S T.S T.S 45° C./ 45 minat 40° C./ 40° C./ 40° C./ Clone 25° C. 25° C. 15 min at 25° C. 25° C.25° C. 25° C. 25° C. Wild type 100 50 32 100 67 100 8 (SEQ ID NO: 1)183H12 109 52 54 118 77 123 28 (SEQ ID NO: 7) 079H6 111 63 66 105 82 11972 (SEQ ID NO: 11) 214A4 111 58 65 101 74 124 38 (SEQ ID NO: 13) 382D8105 86 85 98 93 112 113 (SEQ ID NO: 15) 383A12 93 92 88 96 97 93 92 (SEQID NO: 17) 301C7 94 84 92 96 83 105 89 (SEQ ID NO: 19) 301H3 101 89 7592 78 107 112 (SEQ ID NO: 21)

TABLE 4 Leaf area under normal and increased temperature conditions.Leaf area (%)* Normal growth Increased Clone Line ID conditionsTemperatures Wild type Untransformed 100 ± 13 100 ± 30 Wild type RCA1-1 92 ± 26  62 ± 21 (SEQ ID NO: 1) RCA1-8 106 ± 30  48 ± 16 RCA1-9  88 ±27  45 ± 23 183H12 183H12-2 110 ± 25 131 ± 23 (SEQ ID NO: 7) 183H12-3113 ± 17 142 ± 31 183H12-20 101 ± 30 138 ± 37 382D8 382D8-1 126 ± 23 151± 46 (SEQ ID NO: 15) 382D8-2  90 ± 31  81 ± 32 301C7 301C7-1 105 ± 30101 ± 19 (SEQ ID NO: 19) 301C7-3 104 ± 30 124 ± 31 301C7-7 116 ± 23 115± 36 *The leaf area of the Arabidopsis wild-type untransformed was setto 100%.

TABLE 5 Photosynthesis rates and seed yield under normal and increasedtemperature conditions. Photosynthesis* Seed Clone Line ID (μmol CO₂ m⁻²s⁻¹) yield^(#) (%) Wild type Untransformed 7.85 ± 0.36 72 ± 14 Wild typeRCA1-1 7.25 ± 1.36 74 ± 12 (SEQ ID NO: 1) 183H12 183H12-3 9.41 ± 0.66 87± 8  (SEQ ID NO: 7) 382D8 382D8-1  8.9 ± 0.93 100 ± 11  (SEQ ID NO: 15)301C7 301C7-3 9.03 ± 0.9  87 ± 16 (SEQ ID NO: 19) *Net photosynthesiswas monitored after 2 hours at 30° C. ^(#)Percent of weight of 1000seeds from plants grown under increased temperatures as compared toweight of 1000 seeds from plants grown under normal conditions.

TABLE 6 Seed yield under normal and continuous increased temperatureconditions. 1000-seed weight (mg) Normal growth Continuous increasedClone Line ID conditions temperatures Wild type Untrans- 28.900 ± 4.140 9.484 ± 1.971 formed Wild type RCA1-1 25.837 ± 2.721  8.501 ± 1.921(SEQ ID NO: 1) RCA1-8 21.880 ± 2.839 10.529 ± 0.830 RCA1-9 22.846 ±1.714  9.861 ± 0.596 183H12 183H12-2 19.773 ± 2.112 15.858 ± 3.249 (SEQID NO:7) 183H12-3 25.934 ± 1.869 24.812 ± 1.376 183H12-20 21.730 ± 2.28414.955 ± 2.996

TABLE 7 Germination rates of seeds harvested from plants grown undernormal and continuous increased temperature conditions. Germinated seeds(%) Normal growth Continuous increased Clone Line ID conditionstemperatures Wild type Untrans- 94 ± 2 26 ± 6  formed Wild type RCA1-182 ± 6 4 ± 4 (SEQ ID NO: 1) 183H12 183H12-3 82 ± 2 70 ± 10 (SEQ ID NO:7)

1. An isolated nucleic acid molecule comprising any of SEQ ID NOS: 3, 5,7, 9, 11, 13, 15, 17, 19, 21 or a complement thereof.
 2. An isolatednucleic acid molecule selected from the group consisting of: a. anucleic acid molecule comprising a nucleotide sequence which is at least95% identical to the nucleotide sequence of any of SEQ ID NOS: 3, 5, 7,9, 11, 13, 15, 17, 19, 21 or a complement thereof; b. a nucleic acidmolecule that encodes a polypeptide comprising the amino acid sequenceof any of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22; and c. anucleic acid molecule that hybridizes under stringent conditions with anucleic acid probe consisting of the nucleotide sequence of any of SEQID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or a complement thereof. 3.The isolated nucleic acid molecule of claim 1 or 2, wherein the nucleicacid molecule encodes a polypeptide with increased thermostability ascompared to a polypeptide of SEQ ID NO:
 2. 4. A vector comprising anucleic acid molecule of claim 1 or
 2. 5. The vector of claim 4 that isan expression vector.
 6. A host cell which comprises the vector of claim4.
 7. An isolated polypeptide comprising any one of SEQ ID NOS: 4, 6, 8,10, 12, 14, 16, 18, 20,
 22. 8. An isolated polypeptide selected from thegroup consisting of: a. a polypeptide that is at least 95% identical tothe amino acid sequence of any of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16,18, 20, 22; b. a polypeptide that is encoded by a nucleic acid moleculecomprising a nucleotide sequence that is at least 95% identical to anyone of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or a complementthereof; c. polypeptide encoded by a nucleic acid molecule thathybridizes under stringent conditions with a nucleic acid probeconsisting of the nucleotide sequence of any of SEQ ID NOS: 3, 5, 7, 9,11, 13, 15, 17, 19, 21 or a complement thereof.
 9. The isolatedpolypeptide of claim 7 or 8, wherein the polypeptide has increasedthermostability as compared to a polypeptide of SEQ ID NO:
 2. 10. Atransgenic plant comprising a transgene that expresses a. a polypeptideof claim 8, or b. a nucleic acid molecule of claim
 2. 11. The transgenicplant of claim 10, wherein the plant is selected from the groupconsisting of maize, tomato, potato, rice, soybean, cotton, sunflower,alfalfa, lettuce, canola, sorghum or tobacco plants.
 12. The transgenicplant of claim 10, wherein the transgenic plant has increased heattolerance as compared to a plant that is not transgenic.
 13. A method ofincreasing the heat tolerance in plants comprising expressing apolypeptide of any of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 inthe plant.
 14. The method of claim 13, wherein the polypeptide isexpressed in the one or more plastids of the plant.
 15. The methodaccording to claim 13, wherein the expression of said polypeptideresults in an increase in photosynthesis rate under heated conditions.